Mutated glutathione S-transferase in combination with reduced glutathione shows a synergistic effect in ameliorating oxidative stress and airway inflammation

Oxidative stress is implicated in the pathogenesis of asthma, and antioxidant levels are reduced in asthma patients. Previously, glutathione S-transferase (GST) with reduced IgE binding suppressed oxidative stress and modulated airway inflammation to some extent in mice. GST catalyzes the quenching of reactive oxygen species by reduced glutathione (GSH) and the absence of any one of them may limit antioxidative behavior. This study evaluates the effects of mutated (m) GST with GSH in combination and individually in limiting oxidative stress and inflammatory responses in a mouse model. BALB/c mice were immunized and challenged with ovalbumin. The mice were treated with mGST, GSH, mGST + GSH, or α-lipoic acid by inhalation and sacrificed to evaluate inflammation and oxidative stress parameters. Treatment with the mGST + GSH combination showed significantly reduced total cell (p < 0.01) and eosinophil (p < 0.01) counts in BALF compared to other groups. The lung inflammation score was lowest for the mGST + GSH group, along with reduced IL-4 (p < 0.01) and OVA-specific IgE compared to the other treatment groups. Oxidative stress as per the lipid peroxidation and 8-isoprostane level in BALF of mGST + GSH mice was reduced significantly compared to the individual antioxidants. In conclusion, mGST in combination with GSH has a synergistic effect in reducing airway inflammation compared to the individual antioxidants and has potential for the treatment of asthma.     

Virtual screening using ligand-based pharmacophores for inhibitors of human tyrosyl-DNA phospodiesterase (hTdp1)

Human tyrosyl-DNA phosphodiesterase (hTdp1) inhibitors have become a major area of drug research and structure-based design since they have been shown to work synergistically with camptothecin (CPT) and selectively in cancer cells. The pharmacophore features of 14 hTdp1 inhibitors were used as a filter to screen the ChemNavigator iResearch Library of about 27 million purchasable samples. Docking of the inhibitors and hits obtained from virtual screening was performed into a structural model of hTdp1 based on a high resolution X-ray crystal structure of human Tdp1 in complex with vanadate, DNA and a human topoisomerase I (Top1)-derived peptide (PDB code: 1NOP). We present and discuss in some detail 46 compounds matching the three-dimensional arrangement of the pharmacophoric features. The presented novel chemotypes may provide new scaffolds for developing inhibitors of Tdp1.     

Dihydroindenoisoquinolines function as prodrugs of indenoisoquinolines

Dihydroindenoisoquinolines are analogs of cytotoxic indenoisoquinoline topoisomerase I (Top1) inhibitors, exhibiting potent cytotoxicity but weak inhibitory activity toward Top1. Through COMPARE analysis, cytotoxicity studies in Top1-deficient cells, chemical synthesis and biological evaluation of methylated dihydroindenoisoquinoline 5, we demonstrated that dihydroindenoisoquinolines function as prodrugs of indenoisoquinolines in cancer cells.     

Presence of tear lipocalin and other major proteins in lacrimal fluid of rabbits

The lipocalins are a highly divergent, ubiquitous family of proteins that commonly function in binding lipophilic molecules. Although a specific tear lipocalin is a major component of lacrimal fluid and tears in many mammals, there has been no definitive identification of such a protein in rabbit tears. The goals of this project were to identify the major proteins in rabbit (Oryctolagus cuniculus) lacrimal fluid, so as to determine if they include a lipocalin and, if such a protein is present, to determine its source. Lacrimal fluid was collected from NZW sexually mature female rabbits, and culture medium from rabbit lacrimal gland epithelial (acinar) and interstitial cells was isolated. Proteins from these fluids were separated by SDS-PAGE electrophoresis and analyzed by sequencing the intact proteins and sequencing or mass analysis of fragments derived by trypsin digestion. Proteins of approximately 85 and 67 kDa were identified as rabbit transferrin and serum albumin, respectively, while components of 17 and 7 kDa had N-terminal sequences identical to those of lipophilin CL and AL, respectively. BLAST searches of the nr database with the N-terminal sequence of a protein of 18 kDa did not identify any homologues. However, when used to scan the PROSITE database, it was found to contain a lipocalin signature sequence. It is closely related to two lipocalins previously isolated from rabbit saliva and nasal mucus. Further studies with the N-terminal and internal sequences confirmed that the lacrimal protein is a lipocalin that is truncated at the N-terminus as compared with other tear lipocalins and is more similar to odorant binding proteins from rodents.     

Identification of B cell epitopes of alcohol dehydrogenase allergen of Curvularia lunata

Background/Objective

Epitope identification assists in developing molecules for clinical applications and is useful in defining molecular features of allergens for understanding structure/function relationship. The present study was aimed to identify the B cell epitopes of alcohol dehydrogenase (ADH) allergen from Curvularia lunata using in-silico methods and immunoassay.

Method

B cell epitopes of ADH were predicted by sequence and structure based methods and protein-protein interaction tools while T cell epitopes by inhibitory concentration and binding score methods. The epitopes were superimposed on a three dimensional model of ADH generated by homology modeling and analyzed for antigenic characteristics. Peptides corresponding to predicted epitopes were synthesized and immunoreactivity assessed by ELISA using individual and pooled patients'' sera.

Result

The homology model showed GroES like catalytic domain joined to Rossmann superfamily domain by an alpha helix. Stereochemical quality was confirmed by Procheck which showed 90% residues in most favorable region of Ramachandran plot while Errat gave a quality score of 92.733%. Six B cell (P1–P6) and four T cell (P7–P10) epitopes were predicted by a combination of methods. Peptide P2 (epitope P2) showed E(X)₂GGP(X)₃KKI conserved pattern among allergens of pathogenesis related family. It was predicted as high affinity binder based on electronegativity and low hydrophobicity. The computational methods employed were validated using Bet v 1 and Der p 2 allergens where 67% and 60% of the epitope residues were predicted correctly. Among B cell epitopes, Peptide P2 showed maximum IgE binding with individual and pooled patients'' sera (mean OD 0.604±0.059 and 0.506±0.0035, respectively) followed by P1, P4 and P3 epitopes. All T cell epitopes showed lower IgE binding.

Conclusion

Four B cell epitopes of C. lunata ADH were identified. Peptide P2 can serve as a potential candidate for diagnosis of allergic diseases.     

Determination of dideoxyosone precursors of AGEs in human lens proteins

Dideoxyosones (DDOs) are intermediates in the synthesis of advanced glycation endproducts (AGEs), such as pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of DDOs has not been demonstrated. The aim of this study was to develop a reliable method to detect DDOs in lens proteins. A specific DDO trapping agent, biotinyl-diaminobenzene (3,4-diamino-N-(3-[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]aminopropyl)benzamide) (BDAB) was added during in vitro protein glycation or during protein extraction from human lenses. In vitro glycated human lens protein showed strong reaction in monomeric and polymeric crosslinked proteins by Western blot and ELISA. Glycation of BSA in the presence of BDAB resulted in covalent binding of BDAB to the protein and inhibited pentosidine formation. Mass spectrometric analysis of lysozyme glycated in the presence of BDAB showed the presence of quinoxalines at lysine residues at positions K1, K33, K96, and K116. The ELISA results indicated that cataractous lens proteins contain significantly higher levels of DDO than non-cataractous lenses (101.9±67.8 vs. 31.7±19.5AU/mg protein, p<0.0001). This study provides first direct evidence of DDO presence in human tissue proteins and establishes that AGE crosslink synthesis in the human lens occurs via DDO intermediates.     

Evaluation of inhibitor of apoptosis genes as targets for RNAi-mediated control of insect pests

Apoptosis has been widely studied from mammals to insects. Inhibitor of apoptosis (IAP) protein is a negative regulator of apoptosis. Recent studies suggest that iap genes could be excellent targets for RNA interference (RNAi)-mediated control of insect pests. However, not much is known about iap genes in one of the well-known insect model species, Tribolium castaneum. The orthologues of five iap genes were identified in T. castaneum by searching its genome at NCBI ( https://www.ncbi.nlm.nih.gov/ ) and UniProt ( https://www.uniprot.org/ ) databases using Drosophila melanogaster and Aedes aegypti IAP protein sequences as queries. RNAi assays were performed in T. castaneum cell line (TcA) and larvae. The knockdown of iap1 gene induced a distinct apoptotic phenotype in TcA cells and induced 91% mortality in T. castaneum larvae. Whereas, knockdown of iap5 resulted in a decrease in cell proliferation in TcA cells and developmental defects in T. castaneum larvae which led to 100% mortality. Knockdown of the other three iap genes identified did not cause a significant effect on cells or insects. These data increase our understanding of iap genes in insects and provide opportunities for developing iap1 and iap5 as targets for RNAi-based insect pest control.